HIV-1 Peptides, Nucleic Acids, and Compositions and Uses Thereof

ABSTRACT

This invention features polypeptides, variants thereof, and fragments thereof useful in eliciting an immune response (e.g., neutralizing antibodies) against abroad spectrum of HIV-I isolates. The polypeptides, variants, and fragments include a portion of the gp120 V2 domain of HIV-I. The polypeptides, variants, and fragments display an epitope that is recognized by at least one antibody which neutralizes at least one HIV-I primary isolate. This invention also features nucleic acid sequences encoding those polypeptides. In addition, the invention provides methods of screening for inhibitors of HIV-I entry into cells, as well as methods of treatment using the inhibitors.

CLAIM OF PRIORITY

This application claims benefit of U.S. Provisional Patent ApplicationNo. 60/830,044, filed Jul. 10, 2006, incorporated herein by reference.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

Funds used to support some of the studies disclosed herein were providedby grant numbers AI46283 and AI50452, awarded by the U.S. Public HealthService. Therefore, the U.S. Government may have certain rights to theinvention.

TECHNICAL FIELD

This invention relates to Human Immunodeficiency virus 1 (HIV-1)polypeptides and, in particular, HIV-1 polypeptides useful in elicitingcross-neutralizing antibodies.

BACKGROUND OF THE INVENTION

Despite much progress in recent years towards the characterization offunctional regions of HIV-1 Envelope protein (Env) and in defining majorneutralizing epitopes, progress towards an HIV-1 vaccine capable ofinducing a protective humoral response has been limited (Zolla-Pazner etal. (2004) Nat. Rev. Immunol. 4(3):199-210; Burton et al. (2004) Nat.Rev. Immunol. 5(3):233-236). There is now a realization that typicalantibodies generated in response to infection or immunization withstandard Env proteins do not possess appreciable neutralizing activitiesfor common clinical or primary HIV-1 isolates, even when theseantibodies are directed against neutralization epitopes present in thetarget viruses. One mechanism for this resistance appears to be theshielding of sensitive neutralization domains in intact virions byspecific regions and structural features of HIV-1 clinical isolate SU(HIV-1 SU), located in the V1/V2 region (Pinter et al. (2004) J. Virol.78(10):5205-5215). The highly effective masking of sensitiveneutralization sites by the V1/V2 domain poses a major conundrum forHIV-1 vaccine development. The limited number of known neutralizationtargets that are insensitive to such masking, are poorly immunogenic. Afew exceptional monoclonal antibodies (mAbs) have been identified thatare not sensitive to such effects and possess broad neutralizingactivities for primary isolates. These include the HIV-1 gp120-specificmABs b12 and 2G12 (Burton et al. (2004) Nat. Rev. Immunol.5(3):233-236), and gp41-specific mAbs 2F5 and 4E10 (Zwick et al. (2001)75(22):10892-10905; Kunert et al. (2004) AIDS Res. Hum. Retroviruses20(7):755-762). However, these mAbs possess unusual structures,including unusually large CDR3 regions or have autoreactive properties(Haynes et al., (2005) Science 308(5730):1906-1908), and antibodiesagainst these epitopes are rarely produced in immunized or infectedindividuals.

Thus, it is important to identify additional immunogenic targets thatcan mediate potent neutralization, while also being reasonably wellconserved or present in a limited number of allelic forms that can beformulated into a multivalent vaccine.

SUMMARY OF THE INVENTION

The current invention describes a region in the V2 domain that issemi-conserved, and has been shown in several cases to contain targetepitopes for mAbs that possess very potent neutralizing activities. Theepitopes in this region that determine this activity are referred to asthe V2-Critical Neutralization Domain (V2-CND). The invention describesvariant V2 sequences that express more representative versions of theV2-CND that can induce antibodies that possess potent neutralizingactivities for multiple HIV-1 isolates. This contrasts the generalconsensus in the field that any neutralizing targets in V1 or V2 arehighly type-specific, and limited to a single or at most a very limitednumber of viral sequences, and thus not of any importance in an HIVvaccine {Burton, 2004 #2237; Burton, 2005 #2675; Pantophlet, 2006 #2709;Zolla-Pazner, 2004 #2271}.

Many proteins which include a GP120 V1/V2 of an HIV-1 strain are known,i.e., those disclosed in U.S. Pat. No. 6,815,201, which display anepitope which is recognized by an antibody which neutralizes at leastone HIV-1 primary isolate.

This invention relates to a certain sub-set of such proteins in V2 thatdisplay epitopes that are sensitive neutralization targets. Thesemi-conserved nature of this region suggests that some of these targetsmay be recognized by antibodies that react with multiple viralsequences. Accordingly these targets are of increased importance in anHIV vaccine.

The invention is based in part on the identification of overlappingspecificity determinants of HIV-1 recognized by two highly potentneutralizing antibodies (2909 and C1806g), both of which are present inthe V2 region of HIV-1 Env protein. Analysis of substitution mutationsin the V2 region, using two different HIV-1 isolates, revealed that theminimal epitopes for 2909 and C108g varied from the clade 13 consensussequence only at single positions (position 8 in SEQ ID NO:1 for 2909and position 15 in SEQ ID NO:1 for C108g). Introducing key substitutionsin V2 into both JR-FL and YU2 HIV-1 isolate Env proteins, normallyinsensitive to neutralization by 2909 or C108g, resulted in extremelysensitive neutralization by both antibodies. This showed that the regiondetermining the specificity of 2909 and C108g is very sensitive toneutralization, and that the type-specificity of these antibodies is dueto the presence of rare substitutions at the key positions. Thus thepolypeptide of the invention comprises a sequence that contains aconsensus residue at at least one, and preferably, both of thesepositions.

In addition, it has been found that the neighboring sequences atpositions 9,12,13,and 17-20 of the consensus-containing polypeptide ofthe invention is polymorphic, suggesting the presence of multipleallelic versions of neutralization epitopes in this region.

Accordingly, the epitope-containing polypeptide, (the V2 CriticalNeutralizing Domain, or V2-CND), depicted in SEQ ID NO:1, is believed todescribe multiple variants in this region that are both immunogenic andcapable of mediating the production of antibodies with potent andcross-neutralizing activities against HIV-1.

In one embodiment, the invention relates to an isolated polypeptide,consisting of an amino acid sequence, 30 amino acids in length, and ofsequence EIKNC SFNXT TXXRD KXXXX YXLFY XLDXV (SEQ ID NO:1). The X atposition 9 of SEQ ID NO:1 can be M or I; the X at position 12 of SEQ IDNO:1 can be S, E, G, or N; the X at position 13 can be L, I, or M; the Xat position 17 of SEQ ID NO:1 can be K, R or Q; the X at position 18 ofSEQ ID NO:1 can be K, R, or Q; the X at position 19 of SEQ ID NO:1 canbe K, R or Q; the X at position 20 of SEQ ID NO:1 can be E or V; the Xat position 22 of SEQ ID NO:1 can be S or A; the X at position 26 of SEQID NO:1 can be K or R; and the X at position 29 of SEQ ID NO:1 can be Ior V.

In another embodiment the invention relates to a fragment of the V2-CNDsequence of SEQ ID NO:1. The fragment can contain (a) amino acids 14-20of SEQ ID NO:1 (RDKXXXX), or (b) at least six consecutive amino acids ofSEQ ID NO:1, including at least two of the three residues at positions14-16 and at least two of the X residues at positions 17-20 as definedabove. The V2-CND polypeptide or a fragment thereof can include R, D,and K residues of positions 14-16, and the residues at positions 17-20as depicted by SEQ ID NO:1.

More particularly the V2-CND polypeptide as depicted by SEQ ID NO:1 alsoincludes amino acid sequences where (i) the X at position 17 can be K,the X at position 18 can be Q, the X at position 19 can be K, and the Xat position 20 can be V (KQKV); (ii) the X at position 17 can be K, theX at position 18 can be Q, the X at position 19 can be K, and the X atposition 20 can be E (KQKE); (iii) the X at position 17 can be K, the Xat position 18 can be K, and the X at position 19 can be K (KKK); (iv)the X at position 17 can be K, the X at position 18 can be R, and the Xat position 19 can be Q (KRQ); (v) the X at position 17 can be K, the Xat position 18 can be K, and the X at position 19 can be Q (KKQ); or(vi) the X at position 17 can be K, the X at position 18 can be Q, andthe X at position 19 can be Q (KQQ). The X at position 20 as depicted inSEQ ID NO:1 can be V or E.

The V2-CND polypeptide or a fragment thereof can be a synthetic, orrecombinant molecule, or can be a hybrid polypeptide flanked at one orboth ends of the molecule with either additional sequences that normallyflank this region in gp120, or with heterologous sequences (e.g., oneportion of a polypeptide consisting of the V2-CND polypeptide or afragment of the V2-CND polypeptide and, covalently-linked to it, asecond portion of the polypeptide that is heterologous sequence). Asused herein, “heterologous sequence” refers to any amino acid sequenceoutside of the natural context in which this polypeptide could occur(e.g., HIV-1 gp120 or gp160 polypeptide). This sequence could be, forexample, an antigenic tag (e.g., FLAG, polyhistidine, hemagluttanin(HA), glutathione-S-transferase (GST), or maltose-binding protein(MBP)). Heterologous sequence could also include polypeptides orportions of polypeptides useful in generating or enhancing immuneresponses (e.g., interleukin-2, interleukin-4, interferon gamma, orT-helper sequences), or in the targeting of the polypeptide to specificlocations (e.g., immunoglobulins).

The V2-CND polypeptide or a fragment thereof can also be glycosylated.The glycosylated polypeptide or fragment thereof can be N-glycosylatedat one or both of the N (asparagine) amino acids at positions 4 and 8 ofSEQ ID NO:1. In some embodiments of the V2-CND polypeptide or fragmentthereof, the C residue at position 6 of SEQ ID NO:1 can serve as thefirst member of a disulfide linkage to another macromolecule. Themacromolecule can be another V2-CND polypeptide (e.g., thus resulting ina homodimer) or fragment thereof, or it can be a non-V2-CND polypeptide.This C residue can also be substituted by another residue, such asserine, so that a disulfide bond in not formed at this position.

Yet other embodiments are pharmaceutical compositions containing theV2-CND polypeptide, or a fragment thereof, and a pharmaceuticallyacceptable carrier.

Also provided by the invention is an isolated antibody, orantigen-binding-fragment thereof, that specifically binds to HIV-1 gp120at a sequence that includes SEQ ID NO: 2, and whose binding is dependenton one or more (e.g., 2, 3, 4, 5 or 6) of the amino acid residues atpositions 14-19 of SEQ ID NO:1. The amino acids at positions 17-19 ofSEQ ID NO:1 recognized by the antibody or antigen-binding fragmentthereof can be also be KQKE, KQKV, KKKE, KKKV, KRQE, KRKV, KKQV, orKKQE. Antibody binding can be dependent on the presence of at least twoof the R, D, and K amino acid residues at positions 14-16 of SEQ IDNO:1, and at least two of the amino acid residues at positions 17-19 ofSEQ ID NO:1. Also provided is an isolated antibody or anantigen-binding-fragment thereof that crossblocks the binding of theaforementioned V2-CND-specific antibody or an antigen-binding-fragmentthereof.

The invention also relates to a nucleic acid that encodes the V2-CNDpolypeptides ‘or fragment thereof. Also provided is a vector containinga nucleic acid encoding the V2-CND polypeptides or fragments thereof.The vector can also contain an expression control sequence that isoperably-linked to the nucleic acid. The vector can, optionally, includemore than one V2-CND polypeptide or fragment thereof.

In another embodiment, the invention features a cell containing any ofthe aforementioned nucleic acids or nucleic acid vectors. The cell canbe a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell(e.g., a yeast, insect, or mammalian cell). The cell can also be a humancell.

Also embodied herein are methods of producing a V2-CND polypeptide orfragment thereof. The method involves culturing a cell containing avector, which contains a nucleic acid encoding any of the V2-CNDpolypeptides or fragments thereof described herein, operably-linked toan expression control sequence, under conditions that allow forexpression of the polypeptides or fragments. The method can also includethe step of recovering the polypeptides or fragments from the cellculture. Such recovering from the culture can include recovery from thecells (e.g., from purified cells or cell extracts) or, where a peptideis secreted or otherwise released from a cell, the V2-CND polypeptide orfragments can be recovered from the cell culture media.

Also embodied herein is a method of stimulating the production in amammalian subject of an antibody whose binding in dependent on sequencescontained in the V2-CND and that neutralizes more than one HIV-1 strain.The method includes the step of delivering to a mammalian subject acomposition containing, or consisting of, any of the V2-CND polypeptidesor fragments thereof described herein. The term “delivering” refers toany method (e.g., self-delivery, inhalation, skin contact, or directadministration) by which a subject would come in contact with acomposition described herein. Delivery of the V2-CND-containingpolypeptide or fragment thereof to the mammalian subject can involve theadministration of the composition to the mammalian subject. The methodcan also involve further administering a composition containing one ormore additional V2-CND polypeptides or fragments thereof describedherein to the mammalian subject. As mentioned above, the V2-CNDpolypeptides or their fragments can also contain heterologoussequence(s) at one or both ends. In some aspects, the heterologoussequence can be a single-chain epitope fused to a T-helper sequenceuseful in stimulating an immune response in an individual. T-helpersequences are well known to those of skill in the art and are described,for example, in Zhang et al., (2006) Expert Rev Vaccines 5(2):223-231.The T-helper sequence can be either of the sequences depicted in SEQ IDNO: 3 or SEQ ID NO: 4.

Furthermore, “delivering” can include administering to the subject oneor more vectors, each of which contains a nucleic acid sequence encodingany V2-CND-containing polypeptide or fragment thereof described herein,operably-linked to an expression control sequence. The vector can beadministered as an isolated vector, or a composition containing thevector and a pharmaceutically acceptable carrier. The mammalian subjectcan be a mouse, rat, dog, cat, cow, horse, monkey, or human subject(e.g., a human patient). The compositions for use in the method can befurther delivered to a subject with an immunological adjuvant. Suchadjuvants are well known to those of skill in the art and include, forexample, an aluminum salt or oil in water and an emulsifying agent, anyone of a combination of immunostimulating agents or combinationsthereof. The V2-CND-containing polypeptides, their fragments, nucleicacids encoding the V2-CND polypeptides or their fragments describedherein, and compositions thereof can be administered as compositions ofpolypeptides and/or nucleic acids having one component that elicits animmune response primarily against macrophage-tropic HIV-1 strains and asecond component that elicits an immune response primarily against Tcell tropic HIV-1 strains. It may also be desirable for either or bothcomponents to be composed of a mixture of antigens, e.g., a mixture ofantigens each of which elicits an immune response to a particular HIV-1strain or group of HIV-1 strains.

Also provided is a method of generating a compound that binds to theV2-CND region of HIV-1 gp120. The method includes the steps of (i)designing, based on the 3-dimensional structure of the V2-CNDpolypeptide, a compound containing a region that has the potential tobind to the V2-CND region of HIV-1 gp120; and (ii) synthesizing thecompound. The method can include the additional step of determiningwhether the compound inhibits infection of a cell by at least one HIV-1isolate. Furthermore, featured herein is a method or process ofmanufacturing a compound. The method includes after determining that thegenerated compound that binds to the V2-CND region of HIV-1 gp120inhibits infection of a cell by at least one HIV-1 isolate,manufacturing the compound. This invention also embraces a compoundgenerated and/or manufactured by the above methods.

Also embodied in the invention is a method of inhibiting infection of acell by HIV-1 by contacting a cell and/or an HIV-1 virus with any of theV2-CND polypeptides, fragments or compositions thereof described herein.The method can be an in vitro or an in vivo method. The cell can be amammalian cell (e.g., a mouse, rat, guinea pig, rabbit, dog, cat, horse,goat, cow, monkey, or human cell). The HIV-1 virus can be of any clade,and can be a complete virion, an incomplete virion (e.g., a pseudovirusor empty viral particle).

Another aspect of the invention is a method of treating a subject thatis infected, is suspected of being infected, or is likely to becomeinfected with HIV-1. The method includes delivering to a subject apharmaceutical composition containing any of the V2-CND polypeptides orfragments thereof described herein and a pharmaceutically acceptablecarrier. The subject can be a mammal (e.g., a mouse, rat, cat, dog, cow,horse, or monkey). The subject can be a human (e.g., a human patient).

Also provided herein is a method of identifying a compound that binds tothe V2-CND region of HIV-1 gp120 and neutralizes more than one strain ofHIV-1. The method includes the steps of (i) contacting any of the V2-CNDpolypeptides or fragments thereof described herein with a candidatecompound; and (ii) detecting whether binding of the candidate compoundand the V2-CND polypeptide has occurred. The detecting step can alsoinclude measuring the binding of the candidate compound and the V2-CNDpolypeptide. Candidate compounds that can bind to a V2-CND polypeptidecan include, for example, small molecules, antibodies or antigen-bindingfragments thereof, peptides, peptidomimetics, or aptamers. Thisinvention also encompasses a compound identified by the above method tobe one that binds to the V2-CND region of HIV-1 gp120.

Another aspect of the invention is a method of inhibiting infection of acell by HIV-1. The method includes contacting in vitro a cell and/or anHIV-1 virus with any compound described herein (e.g., a compound thatbinds to the V2-CND region of HIV-1 gp120, or a compound found toinhibit infection of a cell by an HIV-1 virus). The cell can be amammalian cell (e.g., a mouse, rat, cat, dog, cow, horse, monkey, or ahuman cell). The HIV-1 virus can be of any clade, and can be a completevirion or an incomplete virion (e.g., a pseudovirus or an empty viralparticle).

Herein is also provided a method of treating a subject having been, orlikely to have been, infected with an HIV-1 virus. The method includesdelivering to a subject a composition containing a compound that wasdetermined through any of the above described methods to (a) bind to theV2-CND region of HIV-1 gp120 and to (b) inhibit the infection of a cellby HIV-1. The composition of the method can also be delivered with apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier can be a part of the composition or delivered to the subject asa separate composition. The subject can be a mammalian subject,preferably the subject is a human subject (e.g., a human patient).

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. All cited patents, patentapplications, and references (including references to public sequencedatabase entries) are incorporated by reference in their entireties forall purposes.

In case of conflict, the present specification, including definitions,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an alignment of amino acid sequences of the V2 regions ofSF162 and JR-FL HIV-1 viral isolate parental and mutant Envelope (Envs)proteins used to analyze expression of the C108g, 10/76b and 2909epitopes. The HXB2 sequence in included for comparison, and the corepeptide sequence for the C108g and l0/76b epitopes is boxed in. Thepolymorphic residues in this region of the SF162 and JR-FL sequences arehighlighted in bold, and the mutations at these positions required forexpression of these epitopes are indicated. N-linked glycosylation sites(NXT/S) are underlined. SF162 amino acid sequence is SEQ ID NO: 5;SF162(GKV) amino acid sequence is SEQ ID NO: 6; SF162(NI) amino acidsequence is SEQ ID NO: 7; SF162(NI+GKV) amino acid sequence is SEQ IDNO: 8; JR-FL amino acid sequence is SEQ ID NO: 9; JR-FL(GK) amino acidsequence is SEQ ID NO: 10; and HXB2 amino acid sequence is SEQ ID NO:11.

FIGS. 2A, 2B, and 2C are a series of line graphs depictingrepresentative neutralization curves of wild type and chimeric andvariant forms of HIV-1 SF162 and JR-FL viral clinical isolate Envelopeproteins (Envs) with three mAbs that are dependent for binding onsequences in the V2 domain: FIG. 2A: 2909 mAb; Wild-type SF162 gp120(closed squares), JR-FL gp120 with SF162 V1/V2 (open circles), SF162with GKV at positions 167-169 (closed triangles), SF162 with said GKVand NI at positions 161 and 162 (open diamonds), and SF162 with JR-FL V3(open squares). FIG. 2B: C108g mAb; Wild-type SF162 gp120 (closedsquares), SF162 gp120 with NI at positions 161 and 162 (closed circles),SF162 with GKV at positions 167-169 (closed triangles), SF162 with saidGKV at positions 167-169 and NI at positions 161 and 162 (opendiamonds), and Wild-type JR-FL gp120 (closed squares), and JR-FL gp120with GV at positions 167 and 168 (open triangles). Lower Panel, 10/76bmAb; Wild-type SF162 gp120 (closed squares), SF162 gp120 with NI atpositions 161 and 162 (closed circles), SF162 with GKV at positions167-169 (closed circles), SF162 with said GKV and NI at positions 161and 162 (open diamonds), and Wild-type JR-FL gp120 (open triangles), andJR-FL gp120 with GV at positions 167 and 168 (open triangles). Note theexpanded range of antibody concentration for the 2909 titration.Antibody concentrations are presented as ng/ml.

FIG. 3 is an alignment of the amino acid sequences of JR-FL V2 mutantEnv proteins that express the 2909 epitope. Parental HIV-1 virusclinical isolate JR-FL wt is completely resistant to the 2909 antibody.Substitution of the asparagine at 160 with the corresponding lysinepresent in the SF162 sequence did not, alone, confer sensitivity to 2909antibody. However, a double mutant of N160K and K168 resulted in a JR-FLEnv that was more sensitive to 2909 Ab than was SF162 Env. SF162 aminoacid sequence is SEQ ID NO: 12; SF 162(GKV) amino acid sequence is SEQID NO: 13; JR-FL(K160) amino acid sequence is SEQ ID NO: 14; JR-FL(K168)amino acid sequence is SEQ ID NO: 15; JR-FL(SK) amino acid sequence isSEQ ID NO: 16; JR-FL(KK) amino acid sequence is SEQ ID NO: 17.

FIG. 4 is a line graph depicting the neutralization curves of severalJR-FL mutants by mAb 2909. This shows the enhanced sensitivity of theJRFL(KK) mutant to neutralization by 2909. Antibody concentrations arepresented as μg/ml.

FIG. 5 is a line graph depicting the neutralization curves of severalHIV-1 virus JR-FL (N160K/E168K) variants with the 2909 mAb, and severalother standard HIV-1 neutralizing antibodies. Representativeneutralization curves of chimeric JR-FL Env to the 2909 antibody andseveral other standard HIV neutralizing antibodies. Antibodyconcentrations are presented as □g/ml.

DETAILED DESCRIPTION OF THE INVENTION V2-CND Polypeptide

This invention relates to an isolated polypeptide (i.e., the V2-CNDpolypeptide), containing an amino acid sequence, 30 amino acids inlength, and of sequence EIKNC SFNXT TXXRD KXXXX YXLFY XLDXV (SEQ IDNO:1), wherein the X at position 9 of SEQ ID NO:1 is M or I; the X atposition 12 of SEQ ID NO:1 is S, E, G, or N; the X at position 13 of SEQID NO:1 is L, I, or M; the X at positions 17-19 of SEQ ID NO:1 is K, Ror Q; the X at position 20 of SEQ ID NO:1 is E or V; the X at position22 of SEQ ID NO:1 is S or A; the X at position 26 of SEQ ID NO:1 is K orR; and the X at position 29 of SEQ ID NO:1 is I or V. Moreover, theV2-CND polypeptide (SEQ ID NO:1) can also include a polypeptide wherethe X at position 9 of SEQ ID NO:1 is a V, T, or A; the X at position 12of SEQ ID NO:1 is R, D, or E; the X at position 26 of SEQ ID NO:1 is S,T, or N; and the X at position 29 of SEQ ID NO:1 is an L.

The invention also relates to a fragment of the V2-CND polypeptide (SEQID NO:1), wherein the fragment includes amino acids 14-20 of SEQ IDNO:1. Also provided by the invention is a fragment of the V2-CNDpolypeptide that contains at least six consecutive amino acids of SEQ IDNO:1 and includes at least two of the residues at positions 14-16 and atleast two of the X residues at positions 17-20 as defined above. Thefragments can be of any length, shorter than full length V2-CND providedit is at least 6 amino acids in length. In related aspects of theinvention, fragments of the V2-CND polypeptide can include amino acidsequences in which one or more amino acids are deleted and/or added tothe polypeptide

Fragments of the V2-CND polypeptide also include degradation products ofthe polypeptide which may be produced by or in a host cell.

The V2-CND polypeptide, or fragments thereof, of the inventionpreferably has R, D, and K residues of positions 14-16 respectively, andany of the possible residues indicated above for positions 17-20 of SEQID NO:1. In some of these embodiments, the amino acid sequence atpositions 17-19 of SEQ ID NO:1 is KQK, KKK, KRQ, KKQ, or KKQ. In relatedembodiments of the V2-CND polypeptide or fragments thereof, the aminoacid at position 20 of SEQ ID NO:1 is V or E.

The V2-CND polypeptide or a fragment thereof can be synthetic (e.g.,synthesized using a chemical synthesizer, see for example, Miranda etal. (1999) Proc. Natl. Acad. Sci. USA 96:1181-1186). The synthesis ofshort amino acid sequences is well established in the peptide art. See,e.g., Stewart, et al., Solid Phase Peptide Synthesis (2d ed., 1984).

The V2-CND polypeptide or a fragment thereof can also be a recombinantmolecule (e.g., cloned and expressed in bacteria, yeast, or othersuitable host (see below)), and can be hybrid molecules flanked at oneor both ends of the molecule with either additional naturalgp120-derived sequences, or with heterologous sequences (e.g., with oneportion, for example, consisting of the V2-CND polypeptide or a portionof the V2-CND polypeptide, and a second portion encoded by heterologoussequence). Heterologous sequence refers to any amino acid sequenceoutside of the natural context in which this polypeptide could occur(e.g., HIV-1 gp120 or gp160 polypeptide). This sequence could be, forexample, an antigenic tag (e.g., FLAG, polyhistidine, hemagluttanin(HA), glutathione-S-transferase (GST), or maltose-binding protein(MBP)). Heterologous sequence can be of varying length and in some casescould be a larger sequences than the V2-CND polypeptide. Heterologoussequence could also include polypeptides or portions of polypeptidesuseful in generating or enhancing immune responses (e.g., interleukin-2,interleukin-4, interferon gamma, B-cell helper sequences or T-helpersequences), or in the targeting of the polypeptide to specific locations(e.g., immunoglobulins or Fc portions of immunoglobulins). In otheraspects of the invention, the V2-CND polypeptides or fragments thereofcan be linked to secretory, leader sequences, pro- or pre-pro sequences.In addition, heterologous sequences can include a sequence that enhancesthe solubility of the polypeptide, such sequences including, forexample, all or part of an immunoglobulin (IgG) molecule (e.g., theconstant region of an immunoglobulin heavy chain).

The V2-CND polypeptide can be modified by conjugation of additionalfunctional moieties. Examples of such moieties include: biotin,streptavidin, fluorescein, antibodies, fragments, or antigen-bindingfragments thereof. Other moieties embraced by the invention areradiolabels (e.g., ³²P, ³H, ³⁵S, ¹²⁵I) chemical toxins, orchemotherapeutics (e.g., compounds useful in treatment of an HIV-1infection).

In other embodiments, the polypeptides or fragments thereof of theinvention can be also be modified by glycosylation. In particularembodiments of the invention, the glycosylated polypeptides or fragmentsthereof can be N-glycosylated at one or both of the at N amino acids atpositions 4 and 8. Any serine (S), threonine (T), or tyrosine (Y)residues of the polypeptides can also be O-glycosylated. The C residueat position 6 of SEQ ID NO:1 can also serve as one member of a disulfidelinkage to another macromolecule. This additional macromolecule can beanother V2-CND polypeptide or it can be a polypeptide containing aV2-CND polypeptide (e.g., a larger V1/V2 region or the entire gp120protein).

In additional embodiments, the V2-CND polypeptide or its variants orfragments can be modified with moieties that prevent or substantiallyinhibit in vivo degradation (i.e., increase in vivo stability) of thepolypeptides. Examples of such modifications include, withoutlimitation, C-terminal amidation and/or N-terminal acetylation (peptidecapping), or by polyethylene-glycol modification (PEGylation) of theC-terminus (see, for example, Brinckerhoff et al. (1999) Int. J Cancer83(3):326-334). Additional modifications useful in increasing thestability of the V2-CND polypeptide, its variants or fragments thereof,include substituting D-amino acids in place of the natural L-amino acidsat one or more of any residue in the polypeptide. Such methods aredescribed in Tugyi et al. (2005) Proc. Natl. Acad. Sci. USA102(2):413-418 and Hong et al. (1999) Biochem. Pharmacol.58(11):1775-1780.

The V2-CND polypeptide, its fragments, or modified forms thereof, havethe ability of eliciting an immune response in an individual whendelivered or administered (e.g., in eliciting an immune response suchas, a Th1-cell-mediated immunity, or cross-neutralizing antibodiesspecific to (bind to) at least one HIV-1 isolate).

For clarity and understanding regarding the present invention, certainterms are defined below. Additional definitions are set forth throughoutthe detailed description.

A “fragment of the V2-CND polypeptide” as used herein refers to acontiguous portion of the V2-CND polypeptide that is shorter in lengththan the full length V2-CND polypeptide of SEQ ID NO:1 that includes atleast two of the three amino acid residues at residues 14-16 (e.g., R,D, and K at positions 14-16 respectively), and at least two of theresidues at positions 17-20, and is capable of inducing an immuneresponse in a subject. The immune response can be a cell-mediated immuneresponse (e.g., T-cell and mononuclear cell mediated, or T_(H)1response) or a humoral immune response (e.g., a B-cell orantibody-mediated response).

An HIV-1 or human immunodeficiency virus-1, herein refers to an HIV-1virus of any clade (e.g., clade A, AG, B, C, D, E, F, G, H, I, J, K, orL) or from any isolate. Clinical isolates of HIV-1 featured in thisinvention include, for example, HIV-1 JR-FL, HIV-1 YU2, and HIV-1 SF162.

As used herein, the term “treat” or “treatment” is defined as theapplication, delivery, or administration of an agent (e.g., a V2-CNDpolypeptide or a nucleic acid encoding a V2-CND polypeptide or fragmentthereof, or anti-V2-CND antibodies) to a subject (e.g., a humanpatient), or application, delivery, or administration to an isolatedtissue or cell from a subject, e.g., a patient, which is returned to thepatient (see, for example, ex vivo methods below). As used herein,“treatment” can cure, heal, alleviate, relieve, alter, remedy,ameliorate, palliate, improve, or affect the infection or symptoms ofHIV-1 infection in a subject (e.g., a mammal, a human, a human subject).As used herein, a compound or agent that is “therapeutic” is a compoundor agent that causes a complete abolishment of the symptoms of a diseaseor a decrease in the severity of the symptoms of the disease.“Prevention” means that symptoms of the disease (e.g., an HIV-1infection) are essentially absent. As used herein, “prophylaxis” meanscomplete prevention of the symptoms of a disease, a delay in onset ofthe symptoms of a disease, or a lessening in the severity ofsubsequently developed disease symptoms.

As used herein, “vaccine” refers to an agent that induces the immunesystem of the host to respond to the composition or vaccine by producinglarge amounts of CTLs, and/or antibodies specific for the desiredantigen. Consequently, the host typically becomes at least partiallyimmune to later infection, or at least partially resistant to developingan ongoing chronic infection, or derives at least some therapeuticbenefit. In some cases, a vaccine offers complete protective immunity,where protective immunity is the resistance to specific second infectionwith a pathogen that follows a first infection with the pathogen, orprior treatment with a vaccine against the pathogen. A protectiveimmunity, ideally, is complete (100%) prophylaxis (e.g., 100% preventionof a subsequent infection by a pathogen (e.g., HIV-1)), but can also benearly complete (e.g., 70%, 75%, 80%, 85%, 90%, 95%, up to 99%).

As used herein, an amount of a polypeptide, nucleic acid, or ananti-HIV-1 antibody effective to treat a disorder, or a “therapeuticallyeffective amount,” refers to an amount that is effective, upon single ormultiple dose delivery or administration to a subject, in treating asubject with HIV-1. As used herein, an amount of an agent (e.g., aV2-CND polypeptide, a nucleic acid encoding the V2-CND polypeptide, oran anti-V2-CND antibody) effective to prevent or inhibit infection with,and/or disease caused by HIV-1, or a “a prophylactically effectiveamount,” of the antibody refers to an amount which is effective, uponsingle- or multiple dose administration to the subject, in inhibiting ordelaying an infection of a subject by, the occurrence of the onset of,or the recurrence of, HIV-1, or reducing a symptom (e.g., reducing theseverity of a symptom) thereof. “Prophylactic” can also include 100%inhibition of the aforementioned aspects of HIV-1 infection.

In the case of polypeptide sequences that are less than 100% identicalto a reference sequence, the non-identical positions are preferably, butnot necessarily conservative substitutions for the reference sequence.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine and leucine;aspartic acid and glutamic acid; asparagines and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine.

The polypeptide of the invention may also be modified (e.g.,phosphorylation, glycosylation, or disulfide linkage).

The nucleic acid molecules encoding the V2-CND polypeptide of theinvention, herein include cDNA, genomic DNA, synthetic DNA, or RNA, andcan be double-stranded or single-stranded (i.e., either a sense or anantisense strand).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

Nucleic Acids Encoding the V2-CND Polypeptide, Polypeptide Expressionand Purification

Isolated nucleic acids, vectors, and host cell compositions that can beused, e.g., for recombinant expression of the V2-CND polypeptides,variants, and fragments thereof, and for vaccines are provided herein.

Eukaryotic host cells as are known in the art are preferably used forexpression of the V2-CND polypeptides, Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein. Host cells can be any eukaryotic cells (e.g., insect cells,yeast, avian cells (e.g., chicken cells, duck cells), or mammaliancells). Mammalian cells can include cultured cells or a cell line, or aprimate cell such as a Vero cell. Mammalian cells can also include ahuman cell. Other suitable host cells such as plant cells or insectcells are known to those skilled in the art. Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Once the vector or nucleic acid molecule containing the construct(s) hasbeen prepared for expression, the DNA construct(s) may be introducedinto an appropriate host cell by any of a variety of suitable means,i.e., transformation, transfection, conjugation, protoplast fusion,electroporation, particle gun technology, calciumphosphate-precipitation, direct microinjection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene molecule(s) results in the production ofV2-CND polypeptides. This can take place in the transformed cells assuch, or following the induction of these cells to differentiate (forexample, by administration of bromodeoxyuracil to neuroblastoma cells orthe like). A variety of incubation conditions can be used to form thepeptide of the present invention. The most preferred conditions arethose which mimic physiological conditions.

Purification of V2-CND polypeptides. A variety of methodologies known inthe art can be utilized to obtain the polypeptides of the presentinvention. The polypeptides may be purified from tissues or cells whichnaturally produce the peptide. Alternatively, the above-describedisolated nucleic acid fragments could be used to express the V2-CNDpolypeptide in any organism. The V2-CND polypeptides can be purifiedfrom protein extracts or membrane extracts of cells, or preferably fromsupernatant media of cells that express the V2-CND polypeptides insecreted forms.

One skilled in the art can readily follow known methods for isolatingproteins in order to obtain the V2-CND polypeptide fire of naturalcontaminants. These include, but are not limited to: size-exclusionchromatography, HPLC, ion-exchange chromatography, and affinitychromatography (for example, using antibodies specific for sites withinthe polypeptide (i.e., immuno-affinity chromatography), or a ligand thatbinds to an attached tag (e.g., glutathione ligand binding to aglutathione-S-transferase protein tag, or protein A binding to animmunoglobulin Fc domain tag).

Methods of Use of the V2-CND Polypeptide Production of Antibodies

The present invention provides a HIV-1 polypeptide (V2-CND polypeptide),or fragment thereof, for use in the production of antibodies andantigen-binding fragments thereof that bind to the V2-CND polypeptide orfragments thereof. The antibodies can possess HIV-neutralizing activity,and thus be useful both for immunotherapeutic applications or as avaccine.

Antibody Generation

Antibodies or antibody fragments that bind to a V2-CND polypeptide orfragment thereof can be generated by immunization, e.g., using ananimal, or by in vitro methods such as phage display. A polypeptide thatincludes all or part of the V2-CND polypeptide can be used to generatean antibody or antibody fragment. For example, a full length V2-CNDpolypeptide with the following amino acid sequence: EIKNC SFNXT TXXRDKXXXX YXLFY XLDXV (SEQ ID NO:1), wherein the X at position 9 of SEQ IDNO:1 is M or I; the X at position 12 of SEQ ID NO:1 is S, E, G, or N;the X at position 13 of SEQ ID NO:1 is L, I, or M; the X at position 17of SEQ ID NO:1 is K, R or Q; the X at position 18 of SEQ ID NO:1 is K,R, or Q; the X at position 19 of SEQ ID NO:1 is K, R or Q; the X atposition 20 of SEQ ID NO:1 is E or V; the X at position 22 of SEQ IDNO:1 is S or A; the X at position 26 of SEQ ID NO:1 is K or R; and the Xat position 29 of SEQ ID NO:1 is I or V. In addition, the V2-CNDpolypeptide (SEQ ID NO:1) can also include a polypeptide where the X atposition 9 of SEQ ID NO:1 is a V, T, or A; the X at position 12 of SEQID NO:1 is R, D, or E; the X at position 26 of SEQ ID NO:1 is S, T, orN; and the X at position 29 of SEQ ID NO:1 is an L.

In some embodiments, fragments of the V2-CND polypeptide (e.g., carboxy-or amino-terminal truncations, V2-CND polypeptide length of 29-6 aminoacids) can be used as an immunogen to generate antibodies that can bescreened for reactivity to a V2-CND polypeptide. In some embodiments, acell expressing all or part of a V2-CND polypeptide can be used as animmunogen to generate antibodies.

In some embodiments, an immunized animal contains immunoglobulinproducing cells with natural, human, or partially human immunoglobulinloci. In some embodiments, the non-human animal includes at least a partof a human immunoglobulin gene. For example, it is possible to engineermouse strains that are deficient in mouse antibody production andcontain large fragments of the human Ig loci. Using hybridomatechnology, antigen-specific monoclonal antibodies derived from thegenes with the desired specificity can be produced and selected. See,e.g., XenoMouse™, Green et al. Nature Genetics 7:13-21 (1994), US2003-0070185, U.S. Pat. No. 5,789,650, and WO 96/34096.

Non-human antibodies to a V2-CND polypeptide can also be produced, e.g.,in a rodent. The non-human antibody can be humanized, e.g., as describedin U.S. Pat. No. 6,602,503, EP 239 400, U.S. Pat. No. 5,693,761, andU.S. Pat. No. 6,407,213.

Fully human monoclonal antibodies that bind to a V2-CND polypeptide canbe produced, e.g., using in vitro-primed human splenocytes, as describedby Boerner et al., 1991, J. Immunol., 147, 86-95. They may be preparedby repertoire cloning as described by Persson et al., 1991, Proc. Nat.Acad. Sci. USA, 88: 2432-2436 or by Huang and Stollar, 1991, J. Immunol.Methods 141, 227-236; also U.S. Pat. No. 5,798,230. Large nonimmunizedhuman phage display libraries may also be used to isolate high affinityantibodies that can be developed as human therapeutics using standardphage technology (see, e.g., Vaughan et al, 1996; Hoogenboom et al.(1998) Immunotechnology 4:1-20; and Hoogenboom et al. (2000) ImmunolToday 2:371-8; US 2003-0232333). They can also be isolated by cloning ofEBV-immortalized B cells from infected patients, using standardtechniques (Gorny et al., 1989, Proc. Natl. Acad. Sci. USA,86:1624-1628), or recently modified versions of these methods, asdescribed in Traggiai et al., (2004), Nature Med. 10(8):871-875.

In Vivo Methods Uses of V2-CND-Containing Polypeptides to InduceProtective Immunity Administration

V2-CND Polypeptides: The V2-CND polypeptide, fragments thereof, nucleicacids encoding the V2-CND polypeptide and/or fragments of V2-CNDpolypeptides, and compositions comprising the V2-CND polypeptide orencoding nucleic acids (herein referred to as V2-CND agent or agents) ofthe invention are useful in eliciting the production of antibodies whenadministered to a subject (e.g., useful as vaccines or therapeutics).

The V2-CND agents can be administered by a variety of methods known inthe art. For many therapeutic or prophylactic applications, it will beappreciated by the skilled artisan, that the route and/or mode ofadministration will vary depending upon the desired results.

The V2-CND agents may be administered by any conventional methodsincluding aerosol, oral, transdermal, transmucosal, intrapleural,intrathecal, or other suitable routes. Another useful mode ofadministration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). The phrases “parenteral administration”and “administered parenterally” as used herein mean modes ofadministration other than enteral and topical administration, usually byinjection, and include, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural, andintrastemal injection and infusion. For example, the V2-CND agents canbe administered by intravenous infusion or injection. In anotherembodiment, the V2-CND agents are administered by intramuscular orsubcutaneous injection.

The application of V2-CND agents can also be done by way of mucosaladministration (including nasal). Various ways of such administrationare known in the art. The pharmaceutical formulation for mucosaladministration may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other solubilizing or dispersingagents known in the art.

Alternatively, other modes of administration including suppositories maybe desirable.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the subjects to be treated; each unit contains apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

Ideally, the therapeutic or prophylactic regimen can of a single dose ofan appropriate V2-CND agent. However, the therapeutic or prophylacticregimen can include a plurality of doses over a period of time. TheV2-CND agents of the invention can also be combined with additionalappropriate doses of compounds including other epitopes of HIV-1.

Achieving one-dose efficacy can be approached through entrapment ofimmunogen in microparticles. For example, the absorbable suture materialpoly(lactide-co-glycolide) co-polymer can be fashioned intomicroparticles containing immunogen (see, e.g., Eldridge et al. (1991)Molec. Immunol., 28:287-294; Moore et al. (1995) Vaccine 13:1741-1749;and Men et al. (1995) Vaccine, 13:683-689). Following oral or parenteraladministration, microparticle hydrolysis in vivo produces the non-toxicbyproducts, lactic and glycolic acids, and releases immunogen largelyunaltered by the entrapment process. Microparticle formulations can alsoprovide primary and subsequent booster immunizations in a singleadministration by mixing immunogen entrapped microparticles withdifferent release rates. Single dose formulations capable of releasingantigen ranging from less than one week to greater than six months canbe readily achieved.

The V2-CND agents of the invention may also be administered vialiposomes, which serve to target the polypeptides to a particulartissue, such as lymphoid tissue, or targeted selectively to infectedcells, as well as increase the half-life of the peptide composition.Liposomes include emulsions, foams, micelles, insoluble monolayers,liquid crystals, phospholipid dispersions, lamellar layers and the like.In these preparations, the V2-CND agents to be delivered areincorporated as part of a liposome, alone or in conjunction with amolecule which binds to, e.g., a receptor prevalent among lymphoidcells, such as monoclonal antibodies which bind to the CD45 antigen, orwith other therapeutic, prophylactic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired V2-CND agent of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inthe invention are formed from standard vesicle-forming lipids, whichgenerally include neutral and negatively charged phospholipids and asterol, such as cholesterol. The selection of lipids is generally guidedby consideration of, e.g., liposome size, acid lability and stability ofthe liposomes in the blood stream. A variety of methods are availablefor preparing liposomes, as described in, e.g., Szoka et al. (1980) Ann.Rev. Biophys. Bioeng., 9:467, and U.S. Pat. Nos. 4,235,871; 4,501,728;4,837,028; and 5,019,369.

The V2-CND agents of the invention can also be administered ascompositions including a variety of immune-enhancing substances (i.e.,substances that enhance an immune response induced in a subject by aV2-CND agent). For example, V2-CND agents can be co-administered withimmune stimulating complexes (ISCOMS), which are negatively chargedcage-like structure of 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A (saponin). Protective immunity has been generatedin a variety of experimental models of infection including toxoplasmosisand Epstein-Barr virus-induced tumors using ISCOMS as the deliveryvehicle for antigens (see, e.g., Mowat and Donachie, Immunol. Today,23:383-385 (1991)). Immunogenic compositions using ISCOMS are comprisedof the peptides of the invention encapsulated into ISCOMS for delivery.Additional suitable compositions include, for example, lipopeptides(e.g., Vitiello et al., J. Clin. Invest. 95:341 (1995)), peptidecompositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”)microspheres (see, e.g., Eldridge et al., Molec. Immunol. 28:287-94(1991); Alonso et al., Vaccine 12:299-306 (1994); Jones et al., Vaccine13:675-81 (1995)), and multiple antigen peptide systems (MAPs) (see,e.g., Tam, Proc. Natl. Acad. Sci. U.S.A. 85:5409-13 (1988); Tam, J.Immunol. Methods 196:17-32 (1996)). Toxin-targeted deliverytechnologies, also known as receptor-mediated targeting, such as thoseof Avant Immunotherapeutics, Inc. (Needham, Mass.) can also be used.

Alternatively, strategies for increasing the effectiveness of animmunogen (e.g., the V2-CND agents) include, for example, strategieswhereby an immunogenic polypeptide (e.g., the V2-CND polypeptide orfragments of the polypeptide) can be directly modified to enhance theirimmunogenicity or physical properties such as stability. For example,cyclization or circularization of a polypeptide can increase thepolypeptide's antigenic and immunogenic potency.

In addition, single chain fusion proteins comprising the V2-CNDpolypeptide covalently linked to a second sequence (e.g., an HIV-1 V3sequence determinant or a human Ig Fc domain) can also be useful inincreasing the efficacy of the V2-CND agents. Fusion can be obtainedthrough chemical synthesis of a synthetic fusion protein, a chemicallyconjugated fusion of non-synthetic polypeptides, or can be produced byrecombinant techniques (e.g., expressed in a cell from a vectorcontaining a nucleic acid encoding the V2-CND polypeptide fused in framewith a nucleic acid encoding the second sequence). The fusion can bedirect, or include a flexible linker between to the sequences (e.g.,poly-glycine, glycine₄-serine). The V2-CND polypeptides can also becovalently linked to “T-helper epitope” sequences, such as for example,the Pan DR (PADRE) T-helper epitope (see, Pamonsinlapatham P. et al.(2004) Scand. J. of Immunol. 59:504-510) at the amino- orcarboxy-terminus of the V2-CND polypeptide. The V2-CND can also beoligomerized by covalent fusion to oligomerization/multimerizationdomain (e.g., leucine zipper coiled-coil motif of GCN4, or theC-terminal oligomerization domain of p53 protein). Such methods areknown to those of ordinary skill and are also set forth in the Examplesbelow.

The immunogenicity of the polypeptides of the present invention can alsobe modulated by coupling to fatty acid moieties to produce lipidatedpolypeptides. Convenient fatty acid moieties include glycolipid analogs,N-palmityl-S2RS)2,3-bis-(palmitoyloxy)pmopyl-cysteinyl-serine (PAM3Cys-Ser), N-palmityl-S-[2,3 bis (palmitoyloxy)-(2RS)-propyl-[R]-cysteine(TPC) or adipalmityl-lysine moiety. Moreover, the polypeptides may alsobe conjugated to a lipidated amino acid, such as an octadecyl ester ofan aromatic acid, such as tyrosine, including actadecyl-tryrosine (OTH).

The immunogenic agents (e.g., the V2-CND agents), when administered to asubject, also typically include an adjuvant. Adjuvants such asincomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide,alum, Ribi Adjuvant System (RAS), and Titermax (CytRx Corporation, LosAngeles, Calif.) are examples of materials well known in the art (see,for example, Jennings et al. (1995) ILAR Journal 37:119-125). Theproportion of the immunogen (e.g., V2-CND agent) and adjuvant can bevaried over a broad range so long as both are present in effectiveamounts. For example, aluminum hydroxide can be present in an amount ofabout 0.5% of the immunogen mixture (Al₂O₃ basis). On a per-dose basis,the amount of the immunogen can range from about 5 μg to about 100 μμgprotein per patient of about 70 kg. A preferable range is from about 20μμg to about 40 μμg per dose. A suitable dose size is about 0.5 ml.Accordingly, a dose for intramuscular injection, for example, wouldcomprise 0.5 ml containing 20 μg of immunogen in admixture with 0.5%aluminum hydroxide.

Nucleic Acids: The nucleic acids useful for inducing an immune responseinclude at least three components: (1) a V2-CND polypeptide (variant orfragment thereof) coding sequence beginning with a start codon, (2) amammalian transcriptional promoter operatively linked to the codingsequence for expression of the V2-CND polypeptide, and (3) a mammalianpolyadenylation signal operably linked to the coding sequence toterminate transcription driven by the promoter. In this context, a“mammalian” promoter or polyadenylation signal is not necessarily anucleic acid sequence derived from a mammal. For example, it is knownthat mammalian promoters and polyadenylation signals can be derived fromviruses.

The nucleic acid vector can optionally include additional sequences suchas enhancer elements, splicing signals, termination and polyadenylationsignals, viral replicons, and bacterial plasmid sequences. Such vectorscan be produced by methods known in the art. For example, a nucleic acidencoding the desired V2-CND polypeptide or fragments thereof, can beinserted into various commercially available expression vectors. See,e.g., Invitrogen Catalog, 1998. In addition, vectors specificallyconstructed for nucleic acid vaccines are described in Yasutomi et al.(1996) J Virol 70:678-681.

The nucleic acids of the invention can be administered to an individual,or inoculated, in the presence of substances that have the capability ofpromoting nucleic acid uptake or recruiting immune system cells to thesite of the inoculation. For example, nucleic acids encapsulated inmicroparticles have been shown to promote expression of rotaviralproteins from nucleic acid vectors in vivo (U.S. Pat. No. 5,620,896).

A mammal can be inoculated with nucleic acid through any of the routesdescribed herein. The nucleic acids can also be administered, orally, orby particle bombardment using a gene gun. Muscle is a useful tissue forthe delivery and expression of V2-CND polypeptide-encoding nucleicacids, because mammals have a proportionately large muscle mass which isconveniently accessed by direct injection through the skin. Acomparatively large dose of nucleic acid can be deposited into muscle bymultiple and/or repetitive injections. Multiple injections can be usedfor therapy over extended periods of time.

Administration of nucleic acids by conventional particle bombardment canbe used to deliver nucleic acid for expression of a V2-CND polypeptide,variant or fragment thereof, in skin or on a mucosal surface. Particlebombardment can be carried out using commercial devices. For example,the Accell II® (PowderJect® Vaccines, Inc., Middleton, Wis.) particlebombardment device, one of several commercially available “gene guns,”can be employed to deliver nucleic acid-coated gold beads. A Helios GeneGun® (Bio-Rad) can also be used to administer the DNA particles.Information on particle bombardment devices and methods can be found insources including the following: Yang et al. (1990) Proc Natl Acad SciUSA 87:9568 (1990); Yang et al. (1992) CRC Crit Rev Biotechnol 12:335;Richmond et al. (1997) Virology 230:265-274 (1997); Mustafa et al.(1997) Virology 229:269-278; Livingston et al. (1998) Infect Immun66:322-329; and Cheng et al. (1993) Proc Natl Acad Sci USA 90:4455.

The V2-CND polypeptide-encoding nucleic acid can be administered to amucosal surface by a variety of methods including nucleicacid-containing nose-drops, inhalants, suppositories, or microspheres.Alternatively, a nucleic acid vector containing the codon-optimized genecan be encapsulated in poly(lactide-co-glycolide) (PLG) microparticlesby a solvent extraction technique, such as the ones described in Joneset al. (1996) Infect Immun 64:489; and Jones et al. (1997) Vaccine15:814. For example, the nucleic acid is emulsified with PLG dissolvedin dichloromethane, and this water-in-oil emulsion is emulsified withaqueous polyvinyl alcohol (an emulsion stabilizer) to form a(water-in-oil)-in-water double emulsion. This double emulsion is addedto a large quantity of water to dissipate the dichloromethane, whichresults in the microdroplets hardening to form microparticles. Thesemicrodroplets or microparticles are harvested by centrifugation, washedseveral times to remove the polyvinyl alcohol and residual solvent, andfinally lyophilized. The microparticles containing nucleic acid have amean diameter of 0.5 μm. To test for nucleic acid content, themicroparticles are dissolved in 0.1 M NaOH at 100 □C for 10 minutes. TheA₂₆₀ is measured, and the amount of nucleic acid calculated from astandard curve. Incorporation of nucleic acid into microparticles is inthe range of 1.76 g to 2.7 g nucleic acid per milligram PLG.

Microparticles containing about 1 to 100 μg of nucleic acid aresuspended in about 0.1 to 1 ml of 0.1 M sodium bicarbonate, pH 8.5, andorally administered to mice or humans, e.g., by gavage. Regardless ofthe route of administration, an adjuvant can be administered before,during, or after administration of the nucleic acid. An adjuvant canincrease the uptake of the nucleic acid into the cells, increase theexpression of the antigen from the nucleic acid within the cell, induceantigen presenting cells to infiltrate the region of tissue where theantigen is being expressed, or increase the antigen-specific responseprovided by lymphocytes.

Pharmaceutical Compositions

In another aspect, the present invention provides compositions, e.g.,pharmaceutically acceptable compositions. The pharmaceuticalcompositions of the invention can include a “therapeutically effectiveamount” or a “prophylactically effective amount” of a V2-CND agent. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the V2-CNDpolypeptides, nucleic acids, compounds and compositions varies accordingto factors such as the disease state, age, sex, and weight of theindividual, and the ability of the V2-CND polypeptides, nucleic acids,compounds and compositions to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the pharmaceutical composition isoutweighed by the therapeutically beneficial effects. The ability of acompound to inhibit a measurable parameter can be evaluated in an animalmodel system predictive of efficacy in humans. Alternatively, thisproperty of a composition can be evaluated by examining the ability ofthe compound to modulate, such modulation in vitro by assays known tothe skilled practitioner.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result (e.g., complete prevention of the symptoms of adisease, a delay in onset of the symptoms of a disease, or a lesseningin the severity of subsequently developed disease symptoms) against asubsequent challenge by the HIV-1 virus. Typically, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Pharmaceutically acceptable compositions can include the V2-CND agentsdescribed herein, preferably formulated together with a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” means any and all solvents, dispersion media, isotonic andabsorption delaying agents, and the like that are physiologicallycompatible. Useful carriers for immunogenic compositions (e.g., theV2-CND agents of the invention) are well known in the art, and include,for example, thyroglobulin, albumins such as human serum albumin,tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamicacid, influenza, hepatitis B virus core protein, and the like. Thecompositions and vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, typically phosphatebuffered saline. The carrier can be suitable for intravenous,intramuscular, subcutaneous, parenteral, rectal, spinal or epidermaladministration (e.g., by injection or infusion).

The V2-CND agents can be in a variety of forms. These include, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, liposomes and suppositories. The preferred form depends onthe intended mode of administration and therapeutic application. Usefulcompositions are in the form of injectable or infusible solutions.

Compositions typically should be sterile and stable under the conditionsof manufacture and storage. The composition can be formulated as asolution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high concentration of the active ingredient (e.g.,V2-CND agents, e.g., the V2-CND polypeptides, nucleic acids, compoundsor compositions). Sterile injectable solutions can be prepared byincorporating the active compound (i.e., V2-CND agents) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying, both of which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, V2-CND agents can be orally administered, forexample, with an inert diluent or an assimilable edible carrier. TheV2-CND polypeptides agents (and other ingredients, if desired) can alsobe enclosed in a hard or soft shell gelatin capsule, compressed intotablets, or incorporated directly into the subject's diet. For oraladministration, the compounds may be incorporated with excipients andused in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Toadminister a V2-CND polypeptides, nucleic acids, compounds andcompositions of the invention by other than parenteral administration,it may be necessary to coat the V2-CND polypeptides, nucleic acids,compounds and compositions with, or co-administer the V2-CNDpolypeptides, nucleic acids, compounds and compositions with, a materialto prevent its inactivation. Compositions can be administered withmedical devices known in the art.

The following examples are meant to illustrate, not limit, the scope ofthis invention.

EXAMPLES Example 1 Materials and Methods

Monoclonal Antibodies (mAbs)

The monoclonal antibodies in these examples were obtained as follows.C108g was isolated from a chimpanzee infected with the IIIB strain ofHIV-1 and subsequently immunized with isolated MN gp120 (Warrier et al.(1994) J. Virol. 68(7):4636-4642). The characteristics of C108g and itsepitope were previously described (Warrier et al. (1994) J. Virol.68(7):4636-4642; Vijh-Warrier et al. (1995) Mol. Immunol. 32:1081-1092;Wu et al. (1995) J. Virol. 69(4):2271-2278; Pinter et al. (2005) J.Virol. 79(11):6909-6917). 10/76b was isolated from a rat immunized withsoluble HXB10 gp120 (McKeating et al. (1993) J. Virol. 67(8):4932-4944)and characteristics of its epitope have been described (Shotton et al.(1995) J. Virol. 69:222-230). 2909 was isolated from an HIV-1-infectedhuman subject by screening for neutralization of HIV-1 pseudotyped withSF162 Env (Gorny et al. (2005) J. Virol. 79(8):5232-5237).

Chimeric and Variant Forms of HIV-1 Env Protein.

The chimeric and variant forms of HIV-1 Env herein were generated asfollows. The parental SF162 and JR-FL Envs and chimeric forms of theseEnvs in which the V1/V2 domains were exchanged (SF(JR V1/V2)) and JR(SFV1/V2)) were previously described (Pinter et al. (2004) J. Virol.78(10):5205-5215). The various Envs with consensus V3 sequences weregenerated by introducing the necessary modifications sequentially byQuickchange™ site-directed mutagenesis (Stratagene, Inc., La Jolla,Calif.). The same method was used to prepare the V2 variants used tointroduce the C108g and 10/76b epitopes into SF162 Env (Pinter et al.(2005) J. Virol. 79(10:6909-6917) (FIG. 1). These include SF(GKV) inwhich residues N(K)M at position 167-9 were mutated to G(K)V, SF(NI) inwhich residues KV at position 160-1 were mutated to NI, SF(NI+GKV) inwhich both of the above mutations were combined, SF(JR V3) in which theV3 domain corresponded to that of JR-FL, and JR(GK) in which residues DEat position 167-8 were mutated to GK, thereby introducing the C108g and10/76b epitopes.

Viral Neutralization Assays.

Neutralization activity was determined as previously described(Krachmarov et al. (2001) AIDS Res. Hum Retroviruses. 17(18):1737-1748)with a single-cycle infectivity assay using virions generated from theEnv-defective luciferase-expressing pNL4-3.Luc.R⁻E⁻ genome (Connor etal. (1995) J. Virol. 206:935-944) pseudotyped with a molecularly clonedHIV-1 Env of interest. In brief, pseudotyped virions were incubated withserial dilutions of mAbs or polyclonal sera from HIV-infected subjectsfor 1 hour at 37° C., and then added to U87-T4-CCR5 target cells platedout in 96-well plates in RPMI medium containing 10% fetal bovine serum(FBS) and polybrene (10 μg/ml). After 24 hrs, cells were refed with RPMImedium containing 10% FBS and polybrene, followed by an additional 24-48hrs incubation. Luciferase activity was determined 48-72 hrspost-infection with a microliter plate luminometer (HARTA Instruments,Inc., Gaithersburg, Md.), using assay reagents from Promega (Madison,Wis.). ND₅₀ values reported were determined by interpolation fromneutralization curves and are averages of at least three independentassays.

Example 2 Mapping the Critical Determinants of 2909 Reactivity to the V2Domain

The V2 and V3 domain determinants required for 2909 mAb binding weremapped by examining the neutralizing activity of this mAb against aseries of SF162 mutants and variants bearing changes in both the ofthese domains. As previously reported, 2909 possessed extremely potentneutralizing activity for SF162, with an ND₅₀ in the low pg/ml range(0.000058 μg/ml). Substituting the V3 domain of SF162 with that of theclade B consensus sequence (identical to that of the JR-FL isolate)resulted in a significant attenuation in potency, with an almost900-fold increase in ND₅₀. The SF162 sequence differs from that of theclade B consensus at three positions, substitution of T for H at thehighly polymorphic position 13, A for T at position 22 and D for Eatposition 25. An analysis of the effect of single residue substitution atthese three positions on attenuation of neutralization activity of 2909showed that the three changes contributed to this effect to differentextents. The greatest effect (46-fold) was due to the A22T substitution,while the T13H substitution resulted in an 8.3-fold increase and theD25E a 2.3-fold increase in ND₅₀. The overall attenuation matches veryclosely to the product of the three individual effects, and thus theeffects of the individual substitutions on infectivity appeared to bemultiplicative.

2909 also possessed considerable neutralizing activity for a number ofV3 variants that corresponded to the consensus sequences of sixdifferent HIV-1 sub-types sequences, also expressed in the SF162backbone (Table 1). Three variants corresponding to the consensussequences for subtypes C, Al and F required ˜100-fold higher 2909concentrations than SF162 itself to achieve 50% neutralization.Interestingly, these V3 variants were ˜10-fold more sensitive to 2909than the clade B V3 consensus sequence. Other variants corresponding toconsensus sequences for sub-types CFF02_AG and clade H also retainedsensitivity to 2909, but were neutralized less potently than the clade Bconsensus sequence, while the highly variant CRF01_AE consensus sequencewas completely insensitive to neutralization by this mAb.

TABLE 1Effect of V3 sequence variation on neutralization sensitivity of 2909 for chimericSF162 V3 regions

ND₅₀ ^(a) ND₅₀ ratio^(b) SF162   clade C cons

0.000058   0.0048 —   83 clade A1 cons

0.0064 110 clade F cons

0.0076 131 CRF02_AG

0.037 637 clade B cons

0.051 879 clade H cons

0.17 2,931 CRF01_AE cons

>20 >344,000 ^(a)ND₅₀s reported are in μg/ml, and are averages of atleast three independent measurements ^(b)Indicates the fold increase inND₅₀ for the variant V3s over the SF162 sequence. SF162 amino acidsequence is SEQ ID NO: 18; clade C cons. amino acid sequence is SEQ IDNO: 19; Clade A1 cons. amino acid sequence is SEQ ID NO: 20; clade Fcons. amino acid sequence is SEQ ID NO: 21; CRF02_AG amino acid sequenceis SEQ ID NO: 22; clade B cons. amino acid sequence is SEQ ID NO: 23;clade H cons. amino acid sequence is SEQ ID NO: 24; and SRF01_AE cons.amino acid sequence is SEQ ID NO: 25.

While these results confirmed a role for the V3 domain in expression ofthe 2909 epitope and in the extreme sensitivity of SF162 Env to thismAb, they also demonstrated a considerable tolerance of 2909 reactivityfor V3 sequence variation. The neutralizing efficacy for 2909 for manyof the sequences with attenuated reactivity remained in the low ng/mlrange, similar in potency to the best of the standard anti-V3 mAbs andconsiderably more potent than the so-called “broadly-neutralizing”antibodies for these viruses. Thus, while specific features of the SF162V3 sequence are required for the extreme potency of 2909 for viruseswith SF162 Env, 2909 retained sufficient affinity for a number of SF162Env variants expressing V3 domains corresponding to consensus sequencesof multiple viral subtypes to allow 50% neutralization at sub-picomolarconcentrations of the mAb.

Example 3 Mapping the V2 Determinants of 2909 Reactivity

To map determinants in the V2 domain required for 2909 reactivity, theneutralizing activity of 2909 for a series of V1/V2 mutants and variantswas examined. These included a chimeric Env protein in which the V1/V2domain of SF162 Env were replaced by that of JR-FL Env (which is notrecognized by 2909), and V2 domain mutants used to introduce the C108gand 10/76b epitopes into SF162 Env (FIG. 1). Substitution of thecomplete SF162 V1N2 domain by the JR-FL sequence resulted in completeloss of reactivity, confirming a critical role for the V1/V2 domain inthe 2909 epitope (Table 2). Consistent with this, substitution of theSF162 V1/V2 domain into JR-FL Env resulted in neutralization sensitivitysimilar to that of the SF162 chimera with the JR-FL V3 domain,indicating that all of the determinants for the selective reactivity of2909 for SF162 over JR-FL were localized to the V1/V2 and V3 domains.Analysis of V2 mutants that were used to introduce the C108g and 10/76bepitopes into SF162 Env showed a reciprocal relationship between theseepitopes and the 2909 epitope. An essential component of both the C108gand 10/76b epitopes is the presence of residues GKV at position 167-169;for SF162 Env the corresponding residues are NKM (FIG. 1). Substitutionof the G and V into SF162 resulted in a >1,000-fold increase in 2909concentration required for 50% neutralization. An additional componentrequired for expression of the C108g epitope is the N-linkedglycosylation site at position 160. Introducing this site into SF162 bysubstituting the KV present at positions 160-1 with NI, either by itselfor in conjunction with the G-V substitution, resulted in the completeloss of 2909 reactivity (FIG. 2. Table 2). Thus, both of the V2 regionsthat are required for C108g reactivity are also major determinants ofexpression of the 2909 epitope.

TABLE 2 ND_(50S) (μg/ml) for three mAbs dependent on sequences in the V2domain. Env ND₅₀ C108g ND₅₀ 10/76b ND₅₀ 2909 BSF162 >>10 >>10 0.00049SF(JR V1/V2) >>10 >>10 >>10 JR(SF V1/V2) >>10 >>10 0.065 SF(GKV) >>100.032 0.54 SF(NI) >>10 >>10 >>10 SF(NI + GKV) 0.016 3.3 >>10 SF(JRV3) >>10 >>10 0.145 JR(GKV) 0.037 6.0 >>10 JR-FL >>10 >>10 >>10

Example 4 Generating an Enhanced Version of the 2909 Epitope in theJR-FL Env

Additional information about the 2909 epitope was obtained by ananalysis of mutants of JR-FL Env that had additional modifications inthe V2 region defined by the above studies (FIGS. 3 and 4). The parentalJR-FL Env was completely resistant to 2909. Whereas the SF162 sequenceat position 167-9 was NKM, the corresponding sequence in JR-FL was DEV.Changing the E at position 168 to K did not by itself result in 2909reactivity, and neither did introducing a K at position 160. However,combining K168 with changes at position 160 did introduce the 2909epitope. The double mutant in which N160 was changed to S wasneutralized by 2909, although not as potently as the SF162 Env (FIG. 4).However, the JR-FL mutant containing both the K160 and K168 mutationswas considerably more sensitive to neutralization by 2909 than SF162itself. Particularly striking was the ability of 2909 to achieve >99%neutralization of the latter mutant at concentrations as low as 20ng/ml, despite the fact that this Env had a sub-optimal V3 sequence.Eliminating the glycan at position 160 by changing T162 to A did notresult in neutralization by 2909, suggesting that simply removal of theglycan at position 160 was not sufficient for 2909 reactivity, and thatthe presence of lysine residues at both positions 160 and 168significantly enhanced the potency of the epitope for 2909.

A comparison of the sensitivity of the N160K/E168K JR-FL mutant toneutralization by standard mAbs further highlighted the remarkablepotency of 2909 (FIG. 5). 2909 neutralized this virus with an ND₅₀ of<0.04 ng/ml; this was ˜20-fold more potent than IgG-b12, >140-fold morepotent than 2F5, >500-fold more potent than 2G12, and ˜4,000-fold morepotent than a pool of potent V3-specific mAbs. The 160K/168K JR-FLmutant was about 20-fold more sensitive to these anti-V3 mAbs than theparental JR-FL, consistent with a modest masking effect of the 160glycan on the V3 domain. However, the relatively small increase insensitivity to anti-V3 mAbs and the typical sensitivity of this mutantto neutralization by the other mAbs indicates that the changes in V2 didnot introduce a global neutralization sensitivity into this Env. Theseresults provide further support for the exceptional neutralizing potencyof 2909 and the exquisite sensitivity of this target site in V2 toneutralization.

The clade B consensus sequence of the V2 region contains a K at position168, and otherwise is identical to the JR-FL sequence. Thus, the optimal2909 sequence differs from the consensus sequence by only a single aminoacid (K160). Interestingly, the C108g epitope contains the consensussequence at positions 160-2 (NIT), and also differs from the consensussequence by only a single residue, at position 167 (D for G). This showsthat despite the type-specificity of these mAbs, the sequences of bothepitopes are actually highly related to the consensus sequence,differing only by single substitutions at different positions. Thepotent recognition by 2909 of the K160/K168 JR-FL mutant containingconsensus residues DKV at position 167-9 shows that this consensussequence is inherently immunogenic, providing support for the likelihoodthat consensus forms of the C108g epitope would also be immunogenic.

1. An isolated polypeptide, consisting of: (a) an amino acid sequenceEIKNC SFNXT TXXRD KXXXX YXLFY XLDXV (SEQ ID NO:1); or (b) a fragment ofthe sequence, wherein the fragment comprises amino acids 14-20 of SEQ IDNO:1; or (c) a fragment of the sequence, wherein the fragment comprisesat least six consecutive amino acids of SEQ ID NO:1 and includes atleast two of the residues at positions 14-16 and at least two of the Xresidues at positions 17-20, wherein the X at position 9 of SEQ ID NO:1is M or I; the X at position 12 of SEQ ID NO:1 is S, E, G, or N; the Xat position 13 is L, I, or M; the X at position 17 of SEQ ID NO:1 is K,R or Q; the X at position 18 of SEQ ID NO:1 is K, R, or Q; the X atposition 19 of SEQ ID NO:1 is K, R or Q; the X at position 20 of SEQ IDNO:1 is E or V; the X at position 22 of SEQ ID NO:1 is S or A; the X atposition 26 of SEQ ID NO:1 is K or R; and the X at position 29 of SEQ IDNO:1 is I or V.
 2. The polypeptide of claim 1, selected from the groupof a) wherein the X at position 17 is K; the X at position 18 is Q; theX at position 19 is K; and the X at position 20 is V (KQKV, SEQ IDNO:3); b) wherein the X at position 17 is K; the X at position 18 is Q;the X at position 19 is K; and the X at position 20 is E (KQKE, SEQ IDNO:4); c) wherein the X at position 17 is K; the X at position 18 is K;and the X at position 19 is K (KKK); d) wherein the X at position 17 isK; the X at position 18 is R; and the X at position 19 is Q (KRQ); e)wherein the X at position 17 is K; the X at position 18 is K; and the Xat position 19 is Q (KKQ); or f) wherein the X at position 17 is K; theX at position 18 is Q; and the X at position 19 is Q (KQQ).
 3. Anisolated protein comprising the polypeptide of claim 1, and flanked ateither end, or both ends, by a heterologous amino acid sequence.
 4. Theisolated polypeptide of claim 1, wherein the polypeptide isglycosylated.
 5. (canceled)
 6. A pharmaceutical composition comprisingthe polypeptide of claim 1 and a pharmaceutically acceptable carrier. 7.An isolated antibody, or antigen-binding fragment thereof, thatspecifically binds to a polypeptide consisting of SEQ ID NO:6 at anepitope comprising one or more of the amino acid residues at positions14-19 of SEQ ID NO:1.
 8. The isolated antibody of claim 7, wherein theresidues at positions 17-20 of SEQ ID NO:1 are selected from of KQKE(SEQ ID NO:4) or KQKV (SEQ ID NO:3).
 9. An isolated antibody thatcrossblocks the binding of the antibody of claim
 7. 10. The isolatedpolypeptide of claim 1, wherein the polypeptide, when administered to amammalian subject, elicits the production by the subject of an antibodythat has cross-neutralizing activity against HIV-1.
 11. An isolatednucleic acid encoding the polypeptide of claim
 1. 12. A vectorcomprising the nucleic acid sequence of claim
 11. 13. (canceled)
 14. Amethod of stimulating the production of an antibody in a mammaliansubject, the method comprising delivering to a mammalian subject acomposition comprising the polypeptide of claim
 1. 15-19. (canceled) 20.The method of claim 14, further comprising delivering one or moreadditional polypeptides to the subject, wherein each of the one or moreadditional polypeptides is a polypeptide consisting of: (a) an aminoacid sequence EIKNC SFNXT TXXRD KXXXX YXLFY XLDXV (SEQ ID NO:1); or (b)a fragment of the sequence, wherein the fragment comprises amino acids14-20 of SEQ ID NO:1; or (c) a fragment of the sequence, wherein thefragment comprises at least six consecutive amino acids of SEQ ID NO:1and includes at least two of the residues at positions 14-16 and atleast two of the X residues at positions 17-20, wherein the X atposition 9 of SEQ ID NO:1 is M or I; the X at position 12 of SEQ ID NO:1 is S, E, G, or N; the X at position 13 is L, I, or M; the X atposition 17 of SEQ ID NO: 1 is K, R or Q; the X at position 18 of SEQ IDNO: 1 is K, R, or Q; the X at position 19 of SEQ ID NO: 1 is K, R or Q;the X at position 20 of SEQ ID NO: 1 is E or V; the X at position 22 ofSEQ ID NO: 1 is S or A; the X at position 26 of SEQ ID NO: 1 is K or R;and the X at position 29 of SEQ ID NO:1 is I or V.
 21. The method ofclaim 1, wherein the mammalian subject is a human.
 22. The method ofclaim 14, wherein the polypeptide comprises a single-chain epitope fusedto a T-helper amino acid sequence. 23-24. (canceled)
 25. A method ofinhibiting infection of a cell by HIV-1, the method comprisingcontacting a cell or an HIV-1 virus with the polypeptide of claim
 1. 26.(canceled)
 27. A method of treating a subject that is infected, issuspected of being infected, or is likely to be infected with HIV-1, themethod comprising delivering to a subject the pharmaceutical compositionof claim
 6. 28-29. (canceled)
 30. A method of immunizing a human subjectwith an immunogen consisting or containing the polypeptides of claim 1,together with appropriate adjuvants and carriers, in order to induce anantibody response that protects against infection by HIV.
 31. A methodof immunizing a human subject with an immunogen consisting or containingthe polypeptides of claim 1, together with appropriate adjuvants andcarriers, where the specific sequence of the V2-CND polypeptide isidentical or highly related to the sequence of the corresponding regionin a virus or HIV-1 Env gene isolated directly from the same patient.32. An isolated protein comprising the polypeptide of claim 1, andflanked at either end, or both ends, by additional gp120-derived aminoacid sequences that normally flank this region in one or more viralisolates.